Stefania Gagliardi

Selective inhibitors of the osteoclast vacuolar proton ATPase as novel bone antiresorptive agents

The proton ATPase located on the apical membrane of the osteoclast is essential to the bone resorption process. This proton pump is, therefore, an attractive molecular target for the design of novel inhibitors of bone resorption, and potentially useful for the treatment of osteoporosis and related metabolic diseases of bone. Recently, several inhibitors with different degrees of selectivity for the osteoclast V-ATPase have been reported. In particular, systematic chemical modifications of the macrolide antibiotic bafilomycin A1 have identified the minimal structural requirements for activity and allowed the design of simplified analogues that demonstrate high potency and selectivity for the osteoclast enzyme.

Efficient continuous flow synthesis of hydroxamic acids and suberoylanilide hydroxamic acid preparation.

A continuous flow tubing reactor can be used to readily transform methyl or ethyl carboxylic esters into the corresponding hydroxamic acids. Flow rate, reactor volume, and temperature were optimized for the preparation of a small collection of hydroxamic acids. Synthetic advantages were identified as an increased reaction rate and higher product purity. This method was also successfully applied to the multistep preparation of suberoylanilide hydroxamic acid, a potent HDAC inhibitor used in anticancer therapy.

Synthesis of (+)‐Dumetorine and Congeners by Using Flow Chemistry Technologies

An efficient total synthesis of the natural alkaloid (+)-dumetorine by using flow technology is described. The process entailed five separate steps starting from the enantiopure (S)-2-(piperidin-2-yl)ethanol 4 with 29% overall yield. Most of the reactions were carried out by exploiting solvent superheating and by using packed columns of immobilized reagents or scavengers to minimize handling. New protocols for performing classical reactions under continuous flow are disclosed: the ring-closing metathesis reaction with a novel polyethylene glycol-supported Hoveyda catalyst and the unprecedented flow deprotection/Eschweiler-Clarke methylation sequence. The new protocols developed for the synthesis of (+)-dumetorine were applied to the synthesis of its simplified natural congeners (-)-sedamine and (+)-sedridine.

(2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pentadienamide, a novel, potent and selective inhibitor of the osteoclast V-ATPase

Optimisation of a novel series of osteoclast ATPase inhibitors led to (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6- pentamethylpiperidin-4-yl)-2,4-pentadienamide (1) that was the most potent compound in an in vitro osteoclast ATPase assay and in human bone resorption assays. Two of the possible geometric isomers have also been prepared and shown to be significantly less potent than 1.

Replacement of the quinoline system in 2-phenyl-4-quinolinecarboxamide NK-3 receptor antagonists.

Results from a medicinal chemistry approach aimed at replacing the quinoline ring system in the potent and selective human neurokinin-3 (hNK-3) receptor antagonists 1-4 of general formula I are discussed. The data give further insight upon the potential NK-3 pharmacophore. In particular, it is highlighted that both the benzene-condensed ring and the quinoline nitrogen are crucial determinants for optimal binding affinity to the hNK-3 receptor. Some novel compounds maintained part of the binding affinity to the receptor (5, 6, 10 and 13) and compound 5, featuring the naphthalene ring system, appears to be suitable for further modifications; it offers the option to introduce electron-withdrawing groups at position 2 and 4, conferring on the ring an overall electron-deficiency similar to that of the quinoline.

Cinnamic anilides as new mitochondrial permeability transition pore inhibitors endowed with ischemia-reperfusion injury protective effect in vivo.

In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide 22, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction.

Inhibition of Plasmodium falciparum pH regulation by small molecule indole derivatives results in rapid parasite death.

The V-type H+ATPase is critical during the intraerythrocytic stage of the human malaria parasite Plasmodium falciparum. It is responsible for maintaining a near-neutral cytosolic pH (pH 7.3), an acidic digestive vacuole (pH 4.5-5.5) and the generation of an inside-negative plasma membrane potential (approximately -95 mV). Inhibition of this pump is therefore likely to result in profound physiological disturbances within the parasite and parasite death, as illustrated previously by the antiplasmodial activity of the potent and specific inhibitors of the V-type H+-ATPase, bafilomycin A(1) and concanamycin A. In this study we examined the antiplasmodial activity of a series of compounds previously designed, on the basis of the active structural constituents of bafilomycin A(1), to inhibit the osteoclast V-type H+-ATPase. The compounds were tested against up to 4 strains of P. falciparum with varying chloroquine sensitivities. Of the 30 novel compounds tested, 9 had sub-micromolar antiplasmodial IC(50) values, with the most active compound having an IC(50) of 160+/-20 nM. The activity of a number of these compounds was investigated in more detail. We show that these inhibitors acidify the parasite cytosol within seconds and that some inhibitors irreversibly kill the parasite within 0.5-4 h. The antiplasmodial activity of the V-type H+-ATPase inhibitors was strongly correlated with their ability to acidify the parasite cytosol (correlation coefficient 0.98). In combination studies, we show that the inhibitors act indifferently when combined with current antimalarials. Our data support the disruption of parasite pH regulation through inhibition of its V-type H+-ATPase as an antimalarial approach.

Novel bone antiresorptive agents that selectively inhibit the osteoclast V-H+-ATPase

The vacuolar proton pump (V-ATPase) located on the plasma membrane of the osteoclast is a potential molecular target for the discovery of novel bone antiresorptive agents useful for the treatment of osteoporosis. In order to design novel compounds able to selectively inhibit the osteoclast V-ATPase we firstly identified the minimal structural requirements of bafilomycin A1, a macrolide antibiotic which potently inhibits all V-ATPases. This information allowed the design of 2-(indole)pentadienamide derivatives whose optimization led to a novel class of potent inhibitors that demonstrated a high degree of selectivity for the osteoclast V-ATPase. The most interesting derivative, SB-242784, was able to inhibit bone resorption by human osteoclasts in vitro and to completely prevent ovariectomy-induced bone loss in rats when administered orally at 10 mg kg(-1) day(-1). Structure activity relationships of this class of compounds were investigated further by replacing the 2,4-pentadienoyl chain with suitable spacers able to maintain the correct orientation and distance between the indole ring and the amide moiety.

Synthesis and Biological Evaluation of N-Hydroxyphenylacrylamides and N-Hydroxypyridin-2-ylacrylamides as Novel Histone Deacetylase Inhibitors

The histone deacetylases (HDACs) are able to regulate gene expression, and histone deacetylase inhibitors (HDACi) emerged as a new class of agents in the treatment of cancer as well as other human disorders such as neurodegenerative diseases. In the present investigation, we report on the synthesis and biological evaluation of compounds derived from the expansion of a HDAC inhibitor scaffold having N-hydroxy-3-phenyl-2-propenamide and N-hydroxy-3-(pyridin-2-yl)-2-propenamide as core structures and containing a phenyloxopropenyl moiety, either unsubstituted or substituted by a 4-methylpiperazin-1-yl or 4-methylpiperazin-1-ylmethyl group. The compounds were evaluated for their ability to inhibit nuclear HDACs, as well as for their in vitro antiproliferative activity. Moreover, their metabolic stability in microsomes and aqueous solubility were studied and selected compounds were further characterized by in vivo pharmacokinetic experiments. These compounds showed a remarkable stability in vivo, compared to hydroxamic acid HDAC inhibitors that have already entered clinical trials. The representative compound 30b showed in vivo antitumor activity in a human colon carcinoma xenograft model.

New fused benzazepine as selective D3 receptor antagonists. Synthesis and biological evaluation. Part one : [h]-fused tricyclic systems

The synthesis and SAR of a new series of potent and selective dopamine D(3) receptor antagonists is reported. The introduction of a tricyclic [h]-fused benzazepine moiety on the recently disclosed scaffold of 1,2,4-triazol-3-yl-thiopropyl-tetrahydrobenzazepines is reported. A full rat pharmacokinetic characterization is also reported.